Deployment of a Vibrio cholerae ordered transposon mutant library in a quorum-competent genetic background.

Vibrio cholerae, the causative agent of cholera, has sparked seven pandemics in recent centuries, with the current one being the most prolonged. V. cholerae's pathogenesis hinges on its ability to switch between low and high cell density gene regulatory states, enabling transmission between host and the environment. Previously, a transposon mutant library for V. cholerae was created to support investigations aimed toward uncovering the genetic determinants of its pathogenesis. However, subsequent sequencing uncovered a mutation in the gene luxO of the parent strain, rendering mutants unable to exhibit high cell density behaviors. In this study, we used chitin-independent natural transformation to move transposon insertions from these low cell density mutants into a wildtype genomic background. Library transfer was aided by a novel gDNA extraction we developed using thymol, which also showed high lysis-specificity for Vibrio. The resulting Grant Library comprises 3,102 unique transposon mutants, covering 79.8% of V. cholerae's open reading frames. Whole genome sequencing of randomly selected mutants demonstrates 100% precision in transposon transfer to cognate genomic positions of the recipient strain. Notably, in no instance did the luxO mutation transfer into the wildtype background. Our research uncovered density-dependent epistasis in growth on inosine, an immunomodulatory metabolite secreted by gut bacteria that is implicated in enhancing gut barrier functions. Additionally, Grant Library mutants retain the plasmid that enables rapid, scarless genomic editing. In summary, the Grant Library reintroduces organismal relevant genetic contexts absent in the low cell density locked library equivalent.

Clinical Characteristics and Outcomes of SARS-CoV-2 Positive Neonates Born to Persons With SARS-CoV-2 Infection in Pregnancy in Los Angeles County, California, May 22, 2020-February 22, 2021.

BACKGROUND: It is well established that pregnant persons with SARS-CoV-2 are at an increased risk for preterm birth, however, less is known about perinatal outcomes for neonates with intrauterine exposure to SARS-CoV-2. METHODS: Characteristics of 50 SARS-CoV-2 positive neonates born to SARS-CoV-2 pregnant persons positive between May 22, 2020, and February 22, 2021, in Los Angeles County, CA, were assessed. Pattern of neonate SARS-CoV-2 test results and time to positive test was analyzed. Objective clinical severity criteria were applied to assess neonatal disease severity. RESULTS: Median gestational age was 39 weeks with 8 (16%) neonates born preterm. Most (74%) were asymptomatic, while 13 (26%) were symptomatic from any cause. Four (8%) symptomatic neonates met criteria for severe disease, of which 2 (4%) were likely secondary to COVID-19. The other 2 with severe disease had more likely alternate diagnoses, and 1 of these neonates subsequently died at 7 months of life. Among 12 (24%) that were positive within 24 hours after birth, one was persistently positive and represented likely intrauterine transmission. Sixteen (32%) were admitted to the neonatal intensive care unit. CONCLUSION: In this case series of 50 SARS-CoV-2 positive mother-neonate pairs, we found that most neonates were asymptomatic regardless of when they tested positive during the 14 days after birth, that there was relatively low risk of COVID-19 associated severe disease, and that intrauterine transmission can occur in rare cases. Although short-term outcomes are mostly promising, more research is needed to study long-term consequences of SARS-CoV-2 infection in neonates born to positive pregnant persons.

Utilizing Behavioral Science to Improve Antibiotic Prescribing in Rural Urgent Care Settings.

BACKGROUND: Antibiotic-inappropriate prescribing for acute respiratory tract infections (ARTI) is 45% among urgent care centers (UCCs) in the United States. Locally in our UCCs, antibiotic-inappropriate prescribing for ARTI is higher-over 70%. METHODS: We used a quasi-experimental design to implement 3 behavioral interventions targeting antibiotic-inappropriate/non-guideline-concordant prescribing for ARTI at 3 high-volume rural UCCs and analyzed prescribing rates pre- and post-intervention. The 3 interventions were (1) staff/patient education, (2) public commitment, and (3) peer comparison. For peer comparison, providers were sent feedback emails with their prescribing data during the intervention period and a blinded ranking email comparing them with their peers. Providers were categorized as "low prescribers" (ie,  ≤23% antibiotic-inappropriate prescriptions based off the US National Action Plan for Combating Antibiotic Resistant Bacteria 2020 goal) or "high prescribers" (ie,  ≥45%-the national average of antibiotic-inappropriate prescribing for ARTI). An interrupted time series (ITS) analysis compared prescribing for ARTI (the primary outcome) over a 16-month period before the intervention and during the 6-month intervention period, for a total of 22 months, across the 3 UCCs. RESULTS: Fewer antibiotic-inappropriate prescriptions were written during the intervention period (57.7%) compared with the pre-intervention period (72.6%) in the 3 UCCs, resulting in a 14.9% absolute decrease in percentage of antibiotic-inappropriate prescriptions. The ITS analysis revealed that the rate of antibiotic-inappropriate prescribing was statistically significantly different pre-intervention compared with the intervention period (95% confidence interval, -4.59 to -0.59; P = .014). CONCLUSIONS: In this sample of rural UCCs, we reduced antibiotic-inappropriate prescribing for ARTI using 3 behavioral interventions.